• Applied Microscopy
• /
• 제37권2호
• /
• pp.93-102
• /
• 2007

분비백혈구단백분해효소억제제 (SLPI)와 여러 성장인자들은 상처 감염이나 박테리아 침입 시 일어나는 염증반응에 서로 관계가 있지만, 대식세포에서 LPS 자극시 SLPI와 VEGF, bFGF, PDGF 등과 같은 성장 인자들의 발현관계에 대해서는 아직까지 알려진 연구 결과가 없다. 따라서 본 연구는 대식세포 세포주로 알려진 RAW264.7 세포에 SLPI 발현의 적정농도인 LPS에 반응하는 SLPI 및 성장 인자들과 세포외부기질이 발현을 규명하고자 하였다. 역전사효소 중합반응(RT-PCR)과 면역학적 단백질 검출법(Western blotting)은 LPS 처리 후 SLPI와 몇몇 성장인자들 (VEGF, bFGF, PDGF)와 제1형 아교질 mRNA와 SLPI 단백질의 검출을 위해 수행하였다. RAW264.7 세포 주를 mL 당 100 ng의 LPS에 각각 30분, 60분, 90분, 24시간, 48시간동안 노출시켰다. RT-PCR 결과 SLPI mRNA는 시간이 지남에 따라 점점 발현 양이 증가하였고 VEGF와 PDGF mRNA는 초기에 높은 발현 양상을 보였다. 그러나 bFGF와 I형 아교질의 발현은 매우 미약하게 나타났다. SLPI 단백질 발현 역시 mRNA 수준과 마찬가지로 증가하는 양상을 보였는데, 배양액내의 SLPI 단백질양은 전체적으로 감소하는 경향을 보였다. 또한 광학현미경 관찰과 주사전자현미경 관찰결과, LPS가 RAW264.7 세포주의 형태학적인 변화를 일으킴을 확인하였다. 본 결과를 종합하면 SLPI 발현증가의 적정 농도라 생각되는 100ng의 LPS에 의해서 발현되는 VEGF나 PDGF는 SLPI의 발현에 관계가 있는 것으로 생각되지만 추후에 이들 인자들의 단백질이나 유전자 도입을 통하여 발현 관계를 명확히 확인해야 하는 추가실험이 진행되어야 할 것이다.하게 된다.토끼 면역항체를 선모충유충 조직항원에 반응시켰을 때 충체의 표피와 기저층 그리고 EIM 및 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에 황금입자가 표지되었다. 따라서 1일 동안 배설되는 분비배설항원은 선모충 유충의 표피와 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에서 유도되는 반면에 3일 동안 배설되는 분비배설항원은 표피와 stichocyte의 ${\alpha}_0$ 과립에서 유도되고, 선모충유충 감염후 1주, 4주에 실험쥐에서 형성되는 감염항체는 선모충의 표피와 기저층 그리고 EIM에서 분비되는 항원에 의하여 생성된다. 이상의 결과로 선모충의 분비배설항원과 감염항원은 선모충 유충의 표피와 EIM및 stichocyte의 ${\alpha}_0\;{\alpha}_1$ 과립에서 유도되며 이들은 45 kDa 단백을 포함하고 있는 것으로 생각된다.성하고 있는 세포들에는 세포질이 어두운 세포와 밝은 세포가 있었으며, 세포질내에는 전자밀도가 높은 분비과립이 관찰되었다. 전체적인 특징은 눈물샘분비세포 중 장액세포의 것과 비슷하였으나, 과립의 크기는 작았다. 분비관을 구성하는 세포들 사이에도 연접복합체가 매우 잘 발달되어 있었다. 샘포에서 사이관으로 이행되는 곳에서도 샘포세포와 사이관세포 사이에서도 연접복합체가 관찰되었다. 분비관세포의 분비과립 가운데는 중심부분에 전자밀도가 더 높은 중심을 가진 다른 모양의 과립이 관찰되기도 하였다. 의해 사망한 환자는 없었다. 결 론: 자궁경부암 환자에 항암화학요법과 동시에 외부 방사선조사와 고선량률의 강내조사를 시행한 결과 독성이 심하지 않고 국소제어율과 단기 생존율이 양호하여 안전하고 효율적인 치료방법으로 생각된다.

• 한국발생생물학회지:발생과생식
• /
• 제12권2호
• /
• pp.159-168
• /
• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• 한국동물생명공학회지
• /
• 제35권1호
• /
• pp.65-72
• /
• 2020

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

• Biomolecules & Therapeutics
• /
• 제27권1호
• /
• pp.117-125
• /
• 2019

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

• Reproductive and Developmental Biology
• /
• 제32권4호
• /
• pp.263-267
• /
• 2008

This study was conducted to compare the expression pattern of the specific factors associated with pregnancy and angiogenesis during early pregnancy in Hanwoo. Synchronized female Hanwoo ($4{\sim}6$ year-old) were inseminated artificially. After 10 weeks after artificial insemination (AI), the pregnancy was tested by rectal palpation method. Three pregnant and non-pregnant Hanwoo were used in this experiment, respectively. The plasma progesterone level was measured by ELISA. Western blot analysis was performed to detect the expression of pregnancy associated glycoprotein (PAG) or angiogenic factors (VEGF, B-FGF, ANP-1, and TIE-2). The plasma P4 level was increase gradually in pregnant group and maintained high level. The concentration of PAG was significantly higher from $5^{th}$ weeks in pregnant group compared to that of non-pregnant group (p<0.05). The concentrations of the VEGF (p<0.05), B-FGF (p<0.05), and ANP-1 (p<0.05) were significantly increased from $6^{th}\;or\;7^{th}$ week after AI in pregnant group, respectively. And the intensity of TIE-2, ANP-1 receptor, was well matched with ANP-1 (p<0.05). Taken together, it can be postulated that the blood vessels connected with fetus and dam were formed dramatically around 40 days after AI, because the expression levels of the angiogenic factors were increased significantly from this time in pregnant Hanwoo.

• Korean Journal of Acupuncture
• /
• 제34권3호
• /
• pp.146-155
• /
• 2017

Objectives : This study was carried out to investigate the effects of Crataegi Fructus water extract(CFWE) on hair growth in an alopecia model of C57BL/6N mice and human dermal papilla cells(hDPCs). Methods : Six-week old mice were depilated and separated in 3 groups ; CON, MXD(2% Minoxidil), and CFWE. The treatments were applied twice a day for 18 days. The hair growth was determined photographically. The hair density, thickness and length were identified by Folliscope and the weights of body were measured. In dorsal skin tissue, the expression of hair growth-related protein was analyzed by Western blot. In hDPCs with/without $IFN-{\gamma}$, cell proliferation and the expression of hair growth-related genes were analyzed. Results : We observed that CFWE promoted hair growth compared to CON. CFWE improved the hair density, thickness and length compared to CON. CFWE increased the $Wnt/{\beta}$-catenin signaling in dorsal skin. In hDPCs, CFWE accelerated the cell proliferation and inhibited $IFN-{\gamma}$-induced hDPCs degeneration. CFWE increased the mRNA expression of ${\beta}$-catenin, Axin-2, BMP-4, FGF-7, FGF-10, and ALP compared to CON and $IFN-{\gamma}$ treated cells. Conclusions : These results suggest that CFWE has a hair regrowth activity via $Wnt/{\beta}$-catenin signaling and can be useful for the treatment of alopecia.

• 생명과학회지
• /
• 제24권5호
• /
• pp.573-587
• /
• 2014

혈관신생은 모든 조직의 성장과 발달, 그리고 상처회복 등에 매우 중요하다. 지방조직은 우리 몸에서 가장 혈관이 발달된 조직으로서 각 지방세포들은 모세혈관에 둘러싸여 있으며 신생혈관들은 지방세포에 영양분과 산소를 공급한다. 혈관의 내피세포들은 파라크린 신호경로, 세포외 성분, 세포들 간의 직접적인 작용을 통해 지방세포와 교류한다. 활성화된 지방세포들은 VEGF, FGF-2, leptin, HGF와 같은 혈관신생인자들을 생성하며, 이들은 단독으로 혹은 협력하여 혈관신생을 증가시키고 지방조직의 성장과 대사를 촉진한다. 따라서 혈관신생 억제제들은 비만과 비만관련 질환을 치료하는데 유용할 것으로 생각된다.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.80-80
• /
• 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by marked cell death in the striatum and cortex. Stereotaxic injection of quinolinic acid (QA) into striatum results in a degeneration of GABAergic neurons and exhibits abnormal motor behaviors typical of the illness. The objective of this study was carried out to obtain basic information about whether parthenogenetic mouse embryonic stem (PmES) cells are suitable for cell replacement therapy of HD. To establish PmES cell lines, hybrid F1 (C57BL/6xCBA/N) mouse oocytes were treated with 7% ethanol for 5 min and cytochalasin-B for 4 hr to initiate spontaneous cleavage. Thus established PmES cells were induced to differentiate using bFGF (20ng/ml) followed by selection of neuronal precursor cells for 8 days in N2 medium. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days, then a final differentiation step in N2 medium for 7 days. To establish recipient animal models of HD, young adult mice (7 weeks age ICR mice) were lesioned unilaterally with a stereotaxic injection of QA (60 nM) into the striatum and the rotational behavior of the animals was tested using apomorphine (0.1mg/kg, IP) 7 days after the induction of lesion. Animals rotating more than 120 turns per hour were selected and the differentiated PmES cells (1$\times$10$^4$cells/ul) were implanted into striatum. Four weeks after the graft, immunohistochemical studies revealed the presence of cells reactive to anti-NeuN antibody. However, only a slight improvement of motor behavior was observed. By Nissl staining, cell mass resembling tumor was found at the graft site and near cortex which may explain the slight behavioral improvement. Detailed experiment on cell viability, differentiation and migration explanted in vivo is currently being studied.

• Journal of Acupuncture Research
• /
• 제26권2호
• /
• pp.159-170
• /
• 2009

Backgrounds and Objectives: A fracture means a loss of continuity in the substance of bone. Bone differs from other musculoskeletal tissue due to its ability to repair and heal itself without leaving a scar. The cutter head has multinucleated osteoclast cells to resorb the dead bone. The tail, with its conical surface, is lined with osteoblast cells laying down new bone. The conjugation of fracture is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Pyritum, one of the important prescriptions in the oriental medicine, has been used for conjugation fracture. The purpose of this study is to evaluate the effects of administration of Pyritum on activation of osteoblast cells in human body & on tibia bone fracture in mice. Materials and Methods : Four weeks aged 30 female DBA mice were used for this study. They were divided three groups, normal group, control group(fracture elicitate mice: FE group) and experimental group(Pyritum administered mice group after fracture elicitation : PA group). Left tibia bones of mice in FE and PA groups were fractured by bone cutters. MG-63 cells in human body th Pyritum in the ratio of 1 mg/m${\ell}$, and the cells were further incubated for 24 hours. Activation of osteoblast was identified using osteopontin, FGF in vitro test. In vivo test, regeneration of fractured tibia through the morphological changes was observed, and also activation of inflammation through NF-${\kappa}$B p65, iNOS, COX-2, osteoblast through osteopontin, FGF and osteoblast's proliferation in each group was measured. Results and Conclusions : 1. In vitro test for activation of osteoblast cells in human body by Pyritum, osteopontin and FGF production were remarkably increased in Pyritum treated MG-63 cells. 2. In regeneration of fractured tibia by Pyritum, fractured area in external tibia morphology was decreased more in the PA group than that of the FE group. Osteogenesis in fractured area was increased more in the PA group than that of the FE group. Also, endochodrial ossification in central area of fracture and osteoid in lateral area of fracture were increased more in the PA group than those of the FE group. 3. In activation of inflammation by Pyritum administered, activation of NF-${\kappa}$B p65, increase of iNOS and COX-2 production were higher in the PA and the FE groups than those of the control group. Especially, the PA group showed higher activation and increase than those of the FE group. 4. In activation of osteoblast by Pyritum, increase of osteopontin, FGF and osteoblast's proliferation were higher in the PA and the FE groups than those of the control group. Especially, the PA group showed higher increase and proliferation than those of the FE group.

• 한국발생생물학회지:발생과생식
• /
• 제11권3호
• /
• pp.235-243
• /
• 2007

인간 제대혈 세포는 조혈모세포, 중간엽 줄기세포와내피전구세포를 풍부하게 포함하고 있다. 인간 제대혈 속의 중간엽 줄기세포는 조혈모세포와는 달리 다능성 줄기세포이며 신경세포로 분화할 수 있는 잠재성을 가지고 있다. 본 연구에서는 세포배양을 통해 제대혈의 중간엽 줄기세포를 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 배양액에 dimethyl sulphoxide(DMSO)와 butylated hydroxyanisole(BHA)를 첨가하여 유도하였으며 basic fibroblast growth factor(bFGF), retinoic acid(RA), sonic hedgehog(Shh)를 처리하여 콜린성 신경세포로 분화시켰다. DMSO와 BHA에 처리된 중간엽 줄기세포가 빠르게 신경세포 모양으로 분화하는 것을 관찰하였으며, 이것은 면역조직학적 염색에서 신경세포 특이 표지인 $\beta$-tubulin III, 별아교세포에 대한 특이 표지인 GFAP, 희돌기아교세포에 대한 특이 표지인 Gal-C에 대해 양성반응을 나타내었고, 그 비율은 각각 $32.3{\pm}2.9%$, $11.0{\pm}0.9%,\;9.4{\pm}1.0%$였다. RT-PCR 분석에서 배양 단계에 따라 신경세포에 특이적인 표지 인자가 발현됨을 통해, 중간엽 줄기세포가 신경세포로 분화됨을 확인하였다. 또한, 중간엽 줄기세포에 bFGF, RA, Shh를 처리하여 콜린성 신경세포로 분화시켰을 때, 전체 중간엽 세포 중 $31.3{\pm}3.2%$가 신경세포 특이 표지인 $\beta$-tubulin III에 양성반응을 보였으며 이들 세포 중 $70.0{\pm}7.8%$가 콜린성 신경 특이 표지인 ChAT에 양성반응을 보였고, 이것은 Woodbury 방법에 의한 신경분화의 경우보다 3배 가량 높은 비율로 콜린성 신경의 분화를 유도한 것이다. 이러한 실험 결과들은 인간 제대혈의 중간엽 줄기세포가 콜린성 신경세포로 분화가 가능하고 이러한 잠재성을 가진 제대혈 중간엽 줄기세포는 퇴행성 신경질환에 대한 세포 치료제로서 가능성을 제시한다.